The family Enterobacteriaceae includes an array of human, animal and plant pathogens. Human intestinal and extraintestinal infections are caused by specific strains of the closely-related species Escherichia, Shigella, Salmonella and Klebsiella. Although relatively little-studied, Klebsiella pneumoniae nevertheless is a notorious nosocomial pathogen responsible for widespread dissemination of antibiotic resistance. Recently, K. pneumoniae has been identified as the cause of an emerging community-acquired pyogenic liver infection termed KLA (K. pneumoniae liver abscess). This infection is due to specific clones expressing the Hmv (hypermucoviscosity) phenotype, which results from unknown modifications of capsule composition or synthesis. Capsule is an essential pathogenicity factor for K. pneumoniae infections, and Hmv is important for virulence of KLA strains. The Hmv phenotype depends on the auxiliary regulator RmpA, a unique protein that likely forms heterodimers with the global RcsB response regulator. Characterized strains contain multiple rmpA alleles on mobile genetic elements. The relevance to NIAID, and the long-term objectives for this project, are to identify and understand genetic determinants for K. pneumoniae pathogenicity and virulence. The goal of this proposal is to initiate genetic and biochemical analysis in a virulent KLA strain of K. pneumoniae. Studies are designed to identify the functions of RmpA and interacting regulators in the control of virulence factors including capsule and the Hmv phenotype. Research will be in collaboration with a leader in the study of KLA, Prof. Jin-Town Wang at the National Taiwan University College of Medicine. Specific aims will apply a variety of in vivo and in vitro approaches to test five hypotheses: (1a) Duplicate rmpA alleles are at least partially redundant;(1b) RmpA is integrated into the RcsB regulatory network;(2) The RmpA regulon includes virulence genes beyond those involved directly in capsule synthesis and assembly;(3a) RcsB-RmpA heterodimers control capsule biosynthetic operon expression;and (3b) RmpA activity is modulated by phosphorylation. Impacts of the proposed research are: (i) identification and analysis of virulence factors in an emerging pathogen notorious for developing antibiotic resistance;(ii) improved understanding of the unique Hmv phenotype and its control by the RmpA regulon;and (iii) modified concepts regarding global RcsB regulatory network interactions with auxiliary regulators. PUBLIC HEALTH RELEVANCE: Klebsiella pneumoniae, a notorious nosocomial pathogen, has emerged recently as the cause of pyogenic liver abscess in otherwise healthy adults. Hypermucoviscous capsule, a protective layer surrounding the cell surface, is important for this pathology. The public health relevance of this research is to understand a unique regulator that controls hypermucoviscous capsule formation and composition.